The hepatic hemoproteins cytochromes P450 (P450s) are endoplasmic-reticulum (ER)- anchored enzymes engaged in the breakdown of endo- and xenobiotics such as drugs, carcinogens, toxins, natural and chemical products. On exposure to these agents, liver P450 content may be increased due to increased syntheses of its heme and protein moieties, or reduced due to its inactivation/destruction and/or proteolytic degradation. Such drug-mediated modulation of P450 content is known to significantly influence clinical drug-drug interactions. Because P450 synthesis requires heme, defective heme synthesis as in the genetically inherited, acute heme-deficient states clinically known as hepatic porphyrias, can lower P450 levels and thereby impair the metabolism of ingested drugs. Our finding indicates that severe hepatic heme depletion can also profoundly suppress the synthesis of hepatic proteins such as P450s, by shutting off their translation. This stems from increased phosphorylation of the ?-subunit of eukaryotic translational initiation factor eIF2 by a putative hepatic heme-sensitive eIF2? kinase, which is functionally unleashed when hepatic heme is severely depleted. Although the identity of this liver kinase had long remained elusive, we have cloned its cDNA from rat liver and cultured rat hepatocytes, expressed, purified, functionally characterized this enzyme and confirmed its identity as an eIF2? kinase. Our first major aim is to (i) establish its in vivo operation via eIF2?-interactions by various state-of-the-art techniques such as mammalian two-hybrid, chemical crosslinking/proteomic analyses, coimmunoprecipitation, as well as its tissue/intrahepatocellular localization; and (ii) use RNA interference and targeted gene knock out in mice to determine its in vivo physiological and pathological relevance. Our goal is to elucidate the translational suppression that may impair key physiological processes and thus contribute not only to the clinical symptoms of acute hepatic porphyrias, but also influence P450-dependent drug-drug interactions in man. Further, it is now increasingly evident that clinically relevant drug-drug interactions can also result from altered P450 turnover, elicited by drug-mediated P450 stabilization as well as enhanced drug-mediated P450 degradation, such as by the grapefruit furanocoumarins. Such ER-associated degradation of P450s entails their ubiquitylation, extraction from the ER and subsequent proteolysis by the cytosolic 26S proteasome. Heme results in an ER accumulation of ubiquitylated P450s, most likely by blocking their ER extraction and subsequent degradation. ER extraction requires ATP hydrolysis and thus could involve either the p97 AAA ATPase or the proteasomal 19S AAA ATPases. Thus, our second major goal is to characterize the relative roles of p97 and 19S AAA ATPases in this P450 degradation with heme as a probe. These studies are expected to elucidate how heme can affect the birth and death of these P450 proteins and thus modulate the effects and elimination of ingested drugs and environmental agents in man. PUBLIC HEALTH RELEVANCE: Liver cytochromes P450 (P450s) are enzymes engaged in the breakdown of drugs, carcinogens, toxins, natural and chemical agents to water-soluble products. Exposure to these agents can increase liver P450 content or reduce it by enhancing protein degradation and this drug-mediated modulation of P450 content can significantly influence clinical drug-drug interactions. Our studies are aimed at elucidating the role of heme in the cellular processes that control P450 formation as well as their turnover. P450s, like blood hemoglobin, are heme-containing proteins, and their formation in the liver requires heme. However, acute liver heme depletion, a hallmark of acute life-threatening attacks of hepatic porphyrias (clinically defined by genetic defects in heme formation), also blocks P450 formation by functionally activating a normally heme-controlled inhibitor (eIF2? kinase) of protein synthesis, a process we wish to further characterize. Furthermore, heme can also block P450 turnover, a process we propose to mechanistically dissect and further characterize. The P450s chosen as prototypes are orthologs of CYP3A4, the major human liver and intestinal enzyme, and 2 other liver P450s that together are responsible for the metabolism of approximately 75% of clinically relevant drugs, toxins, and carcinogens, with consequently significant potential for drug-drug interactions and toxicity. [unreadable] [unreadable] [unreadable] [unreadable]